Much research on immune senescence has been focused on T-cells, mainly because a low numbers of naive T-cells in the peripheral blood are the first sign of immune senescence [1]. Furthermore, changes in the T cell receptor (TCR) repertoire diversity have been linked to aging and immune senescence [2]. Aside from the natural reduction in T-cell renewal and TCR repertoire diversity, cytomegalovirus (CMV) infection has a profound influence on subset distribution, phenotype and potentially also on the function of T cells in the elderly [3-5]. However, it is still unclear whether CMV infection is the driver of T cell immune senescence and if so, to what extent. Despite these interesting and suggestive observations, lacking proper tools that enable the systematic study of the rare CMV specific T cells in CMV negative individuals at both the sequence and functional level hinders the quest for an answer to the above questions. Therefore, we propose to develop: 1) a next-generation sequencing based technology to analyze the TCR sequence repertoire;2) a tetramer staining and isolation method to examine the TCR functional repertoire;and 3) a microfluidic chip-based single cell quantitative PCR (qPCR) technology to dissect functional capabilities of CMV specific T cells. We will apply these technologies to follow and characterize specific T cell populations in older and younger blood donors and correlate this with the timing of CMV seroconversions in both groups. We hypothesize that due to abnormalities in the global and CMV specific T cell repertoires (holes and/or reduced frequency and/or diversity in both TCR sequence and ligand repertoire) and/or defects in the functional capacities of CMV specific precursor T cells, the elderly are predisposed to CMV infection. Furthermore, prolonged period of an inefficient immune response to the virus may result in desensitized immune responses that further drive the development of immune senescence. Our specific aims are: Aim 1. Study global TCR 1 and 2 chain gene diversity difference in young and elderly cohorts using high-throughput sequencing Aim 2. Correlate T cell receptor sequence repertoire with ligand repertoire and phenotype in T cells specific for CMV immunodominant epitopes isolated from young and elderly cohorts Aim 3. Compare functional capabilities of T cells specific for CMV epitopes isolated from young and elderly cohorts PUBLIC HEALTH RELEVANCE: Data generated from this study will help us understand how the TCR repertoire ages and what is the relation between CMV infection and immune senescence. Technologies developed here will be useful to advance clinical diagnostics in CMV infection and immune senescence.